We have previously reported that pancreatic islet (-cells and clonal HIT insulinoma cells express an ATP-stimulatable Ca2+-independent phospholipase A2 (ASCI-PLA2) enzyme and that activation of this enzyme appears to participate in glucose-stimulated insulin secretion. To further examine this hypothesis, glucose-responsivity and expression of ASCI-PLA2 activity in various insulinoma cell lines were examined. Secretagogue-stimulated insulin secretion was observed with (TC6-f7 and early passage (EP)- (TC6 cells. In contrast, RIN-m5f, BTC3, and late passage (LP)- (TC6 cells exhibited little secretagogue-induced secretion. These and previous results suggest that the ATP-stimulatable PLA2 enzyme may participate in glucose-induced insulin secretion. Mass spectrometric analysis of insulinoma cell phospholipid content revealed a decline in arachidonate-containing species during several passage in cultures that correlated with reduced glucose-responsivity of insulin secretion and with reduced membrane associated, Ca2+-independent PLA2 activity.